Cells continuously take up nutrients and signaling molecules. Internalized proteins destined for degradation are routed from the plasma membrane through early endosomes, multivesicular bodies (MVBs), and finally to lysosomes. In specialized metazoan cells, endosomes also serve as intermediates in protein trafficking to lysosome-related organelles including melanosomes in the skin and eye, lamellar bodies in type II lung cells, dense granules in platelets, and lytic granules in cytotoxic T-cells. Genetic defects that interfere with trafficking in this pathway are the underlying causes of several disorders including Chediak-Higashi and Hermansky-Pudlak syndrome (HPS). The Drosophila compound eye is an excellent model system for a genetic analysis of endocytic trafficking. Mutations in many genes necessary for this process have been identified as eye color mutations because they interfere with the delivery of biosynthetic cargo to pigment granules. In the developing eye, the ligands Boss and Delta provide direct assays to analyze the contribution of genes to endocytic trafficking in vivo. This proposal is aimed at exploiting the features of the Drosophila eye for a genetic dissection of endocytic trafficking in multicellular organisms. In Aim 1, we plan to dissect the specific roles each of two alternative Vps-C complexes have in regulating endocytic trafficking, and the biogenesis of lysosome-related organelles. For this purpose, we will use loss-of-function models of the two pairs of alternative subunits Car/dVps33A, dVps16A, dVpsSSB, and dVps16B. In Aim 2, we will determine the specific step in endocytic trafficking that requires red, one of the genes discovered in a large-scale screen for HPS-related mutations. In Aim 3, we will dissect how the dual functions of Drosophila Acinus in membrane trafficking and chromatin condensation are regulated. In Aim 4, we will analyze the function of dPallidin in the delivery of cargo to lysosome-related organelles. We have generated a dpallidin null allele to test two alternative models that suggest that Pallidin may regulate early endosomal fusion or exit from MVBs. [unreadable] [unreadable] [unreadable]